behavioral/systems/cognitive ... · based transformation vector containing a 13.2 kb genomic dper...

Behavioral/Systems/Cognitive

Two Distinct Modes of PERIOD Recruitment onto dCLOCKReveal a Novel Role for TIMELESS in Circadian Transcription

Woo Chul Sun,1* Eun Hee Jeong,1* Hyun-Jeong Jeong,1 Hyuk Wan Ko,2 Isaac Edery,3 and Eun Young Kim1

1Neuroscience Graduate Program, Institute for Medical Sciences, Ajou University School of Medicine, San5, Wonchon-dong, Suwon, Kyunggi-do, 443-721,Republic of Korea, 2Age-Related and Brain Diseases Research Center, Neurodegeneration Control Research Center, School of Medicine, Kyung HeeUniversity, Hoegi-dong 1, Dongdaemun-Gu, Seoul, 130-701, Republic of Korea, and 3Department of Molecular Biology and Biochemistry, RutgersUniversity, Center for Advanced Biotechnology and Medicine, Piscataway, New Jersey 08854

Negative transcriptional feedback loops are a core feature of eukaryotic circadian clocks and are based on rhythmic interactions betweenclock-specific repressors and transcription factors. In Drosophila, the repression of dCLOCK (dCLK)-CYCLE (CYC) transcriptionalactivity by dPERIOD (dPER) is critical for driving circadian gene expression. Although growing lines of evidence indicate that circadianrepressors such as dPER function, at least partly, as molecular bridges that facilitate timely interactions between other regulatory factorsand core clock transcription factors, how dPER interacts with dCLK-CYC to promote repression is not known. Here, we identified a smallconserved region on dPER required for binding to dCLK, termed CBD (for dCLK binding domain). In the absence of the CBD, dPER isunable to stably associate with dCLK and inhibit the transcriptional activity of dCLK-CYC in a simplified cell culture system. CBD issituated in close proximity to a region that interacts with other regulatory factors such as the DOUBLETIME kinase, suggesting thatcomplex architectural constraints need to be met to assemble repressor complexes. Surprisingly, when dPER missing the CBD(dPER(�CBD)) was evaluated in flies the clock mechanism was operational, albeit with longer periods. Intriguingly, the interactionbetween dPER(�CBD) and dCLK is TIM-dependent and modulated by light, revealing a novel and unanticipated in vivo role for TIM incircadian transcription. Finally, dPER(�CBD) does not provoke the daily hyperphosphorylation of dCLK, indicating that direct interac-tions between dPER and dCLK are necessary for the dCLK phosphorylation program but are not required for other aspects of dCLKregulation.

IntroductionCircadian (�24 h) rhythms are driven by cell autonomous clocksthat are generally composed of interconnected transcriptionaland translational feedback loops (Dunlap, 1999). Studies usingDrosophila have made seminal contributions to our understand-ing of clock mechanisms in general and those of animals in par-ticular (Edery, 2000; Allada et al., 2001). In Drosophila, the majortranscriptional negative feedback loop is comprised of dCLOCK(dCLK) and CYCLE (CYC), that heterodimerize to activate thedaily transcription of target genes, including the core clock genesperiod (dper) and timeless (tim), whose protein products partici-pate in the repression of dCLK-CYC-mediated gene expression(Hardin, 2006). dPER and TIM interact in the cytoplasm, anevent that promotes their nuclear entry (Vosshall et al., 1994;Myers et al., 1996; Saez and Young, 1996; Meyer et al., 2006),where it is thought that the binding of dPER to dCLK is a crucial

step in blocking the transcriptional activity of the dCLK-CYCcomplex (Darlington et al., 1998; Lee et al., 1998, 1999; Bae et al.,2000). More recent evidence indicates that dPER does not di-rectly inhibit dCLK-CYC-mediated transcription but likely func-tions as a scaffold to promote the interaction between largelyuncharacterized inhibitory factors and dCLK-CYC (Kim andEdery, 2006; Yu and Hardin, 2006; Yu et al., 2006, 2009; Kim etal., 2007; Chen et al., 2009).

How TIM contributes to repressing dCLK-CYC activity ismore enigmatic but is mainly thought to be primarily due to itseffects on the subcellular localization and stability of dPER. Inaddition to stimulating the nuclear entry of dPER, TIM also actsto stabilize dPER in the cytoplasm and nucleus by attenuating theability of the DOUBLETIME (DBT; Drosophila homolog ofCK1�/�) kinase to evoke the rapid degradation of dPER (Price etal., 1995, 1998; Kloss et al., 1998, 2001; Ko et al., 2002). Theobservation that TIM is present in a complex with dCLK-CYCduring the night while peak activity of transcriptional inhibitionoccurs (Lee et al., 1998) raised the possibility that TIM might havea more direct role as a repressor. However, dPER has been shownto inhibit dCLK-CYC-mediated transcription independent ofTIM in vitro and in vivo (Rothenfluh et al., 2000; Ashmore et al.,2003; Chang and Reppert, 2003), casting doubt on a direct phys-iological role for TIM in transcriptional repression.

To better understand how dPER inhibits the transactivationpotential of dCLK-CYC, we identified a small conserved region of

Received May 8, 2010; revised Aug. 31, 2010; accepted Sept. 6, 2010.This work was supported by Basic Science Research Program (2008-0058506, NRF-2008-521-C00220) and

Chronic Inflammatory Disease Research Center (R13-2003-019) through the National Research Foundation of Korea(NRF) funded by the Ministry of Education, Science and Technology. The work was also supported by a NationalInstitutes of Health grant to I.E. (NS03958). We thank Dr. Blau for kindly providing monoclonal anti-PDF antibody.

*W.C.S. and E.H.J. contributed equally.Correspondence should be addressed to Eun Young Kim, Institute for Medical Sciences, Ajou University School of

Medicine, San5, Wonchon-dong, Suwon, Kyunggi-do, 443-721, Republic of Korea. E-mail: [email protected]:10.1523/JNEUROSCI.2366-10.2010

Copyright © 2010 the authors 0270-6474/10/3014458-12$15.00/0

14458 • The Journal of Neuroscience, October 27, 2010 • 30(43):14458 –14469

dPER required for its binding to dCLK, termed CBD (for dCLKbinding domain). dPER missing the CBD (dPER(�CBD)) is un-able to inhibit the transcriptional activity of dCLK-CYC in a sim-plified cell culture system. Surprisingly, when dPER(�CBD) wasevaluated in flies the clock mechanism was very robust, althoughit did have a longer period. Intriguingly, despite the inability ofdPER(�CBD) to directly bind dCLK, it was still associated withdCLK. We show that this association is TIM-dependent andmodulated by light, revealing a novel and unanticipated role forTIM in regulating circadian transcription.

Materials and MethodsPlasmids and methods for S2 cell-based assays. The pAct-dper, pAct-tim,pAct-dper-V5, pMT-dClk-V5, pMT-dbt-V5, and pAct-tim-3HA plas-mids were described previously (Ceriani et al., 1999; Ko et al., 2002; Kimand Edery, 2006; Kim et al., 2007). pAct-dper�C3-V5, pAct-dper�NC3-V5, pAct-dper�C4-V5, and pAct-dper�NC4-V5 (Fig. 1 A) were gener-ated by site-directed mutagenesis using the QuikChange Site-Directed

Mutagenesis Kit (Stratagene). All final constructs were verified by DNAsequencing.

S2 cells were obtained from Invitrogen and transfected using effectenefollowing the manufacturer’s protocol (Qiagen). dCLK-dependenttransactivation using a luciferase (luc) reporter assay was performed asdescribed previously (Darlington et al., 1998), with slight modifications(Chang and Reppert, 2003; Kim and Edery, 2006). Briefly, S2 cells wereplaced in 24-well plates and cotransfected with 10 –20 ng of control pAct-dper and/or different modified versions of pAct-dper plasmids, alongwith 10 ng of perEluc, 30 ng of pAct-�-gal-V5/His, and 2 ng of pMT-dClk-V5. In some experiments, 20 –100 ng of pAct-tim plasmids werecotransfected, as indicated. One day after transfection, dClk expressionwas induced with 500 �M CuSO4 (final in the media), and after anotherday cells were washed in PBS, followed by lysis in 300 �l of ReporterLysis Buffer (Promega). Aliquots of cell extracts were assayed for�-galactosidase and luciferase activities using the Luciferase AssaySystem and protocols supplied by the manufacturer (Promega).

Fly strains and behavioral assays. To generate transgenic flies that pro-duce the dPER�CBD protein we used a previously described CaSpeR-4-

Figure 1. dPER(�C4) is defective in transcriptional repression and DBT-dependent progressive phosphorylation in S2 cells. A, Schematic diagram of functional domains in the C-terminal half ofdPER protein (accession no. P07633). Horizontal lines denote regions internally deleted from full-length dPER to generate mutants analyzed in this study. Domains are as follows: Thr-Gly repeats(dark gray box with label TG); dCLK:CYC inhibitory domain (CCID; amino acids 764-1034; white box) (Chang and Reppert, 2003); dPER DBT binding domain (dPDBD; amino acids 755– 809; bracket)(Kim et al., 2007); putative nuclear localization sequence (amino acids 813-840, hatched box) (Chang and Reppert, 2003). B, S2 cells were transiently transfected in the presence (�) or absence of(�) pMT-dClk-V5. In addition, some cells were cotransfected with different versions of pAct-dper, as indicated. Shown are the average values from three independent experiments for relativeluciferase activity. Luc activity in the absence of pMT-dClk-V5 was set to 1, and all other values were normalized. C–E, S2 cells were transiently transfected with different versions of V5-taggedpAct-dper (C, E) or nontagged pAct-dper (D), as indicated (top of panels). In addition, some cells were cotransfected with pMT-dbt-V5, as indicated (�). Exogenous DBT was induced 36 h aftertransfection by adding 500 �M CuSO4 to the medium. Cells were harvested at the indicated times (C), 24 h (D), or 36 h (E) after induction, and protein extracts were either directly analyzed byimmunoblotting (C–E) or first subjected to immunoprecipitation in the presence of anti-V5 antibodies (D, top). Arrowheads indicate hypophosphorylated isoforms in each variants of dPER.D, “Input” indicates protein lysates used for immunoprecipitation. dPER was visualized with anti-dPER antibodies, whereas recombinant DBT with anti-V5 antibodies.

Sun et al. • dCLK Binding Domain on dPER Protein J. Neurosci., October 27, 2010 • 30(43):14458 –14469 • 14459

based transformation vector containing a 13.2 kb genomic dper insertthat was modified with sequences encoding for the HA epitope tag and astretch of histidine residues just upstream of the dper translation stopsignal, termed 13.2( per�-HA10His) (Lee et al., 1998). Deletion of se-quences encoding amino acids 926 –977 from dPER was performed usingthe QuikChange Site-Directed Mutagenesis Kit (Stratagene) with an ap-propriate dper genomic subfragment, confirmed by DNA sequencingand reconstructed into the above mentioned transformation vector toyield 13.2( per�CBD-HA10His) (herein referred to as dper(�CBD)).Transgenic flies were generated by BestGene Inc. using standard P element-mediated transformation techniques and w1118 embryos as hosts. Two in-dependent germ-line transformants bearing the dper(�CBD) transgene in aper� background were obtained and then crossed into a wper01 geneticbackground to yield wper01;p{dper(�CBD)}-HAHis (herein referred to aswper01;p{dper(�CBD)}). Generation of transgenic flies expressing a wild-type version of the recombinant dPER protein was described in a previousreport (Kim et al., 2007), and one of the lines (M16) in a wper01 geneticbackground (herein referred to as wper01;p{dper(WT)}) was used as controltransgenic flies.

The locomotor activities of individual flies were measured as pre-viously described using the monitoring system from Trikinetics(Hamblen-Coyle et al., 1992). Briefly, young adult flies were used for theanalysis and kept in incubators at 25°C, exposed to at least 4 d of 12 h lightfollowed by 12 h dark [12:12LD; where zeitgeber time 0 (ZT0) is definedas the time when the light phase begins] and subsequently kept in con-stant dark conditions for 5– 8 d. The locomotor activity data for eachindividual fly was analyzed using the FaasX software (Fly Activity Anal-ysis Suite for MacOSX), which was generously provided by F. Rouyer(Centre National de la Recherche Scientifique, Paris, France). Periodswere calculated for each individual fly using � 2 periodogram analysis andpooled to obtain a group average for each independent transgenic line orgenotype. Power is a measure of the relative strength of the rhythmduring DD. Individual flies with a power �10 and a “width” value of 2 ormore (denotes number of peaks in 30 min increments above the perio-dogram 95% confidence line) were considered rhythmic.

Immunoblotting and immunoprecipitation. Protein extracts from S2cells were prepared as previously described (Kim et al., 2007). Briefly, thecells were lysed using modified-RIPA (radioimmunoprecipitation assay)buffer (50 mM Tris-HCl, pH7.5, 150 mM NaCl, 1% NP-40, 0.25% sodiumdeoxycholate) with the addition of Protease Inhibitor Cocktail (Roche)and PhosSTOP (Roche). For detection of recombinant dCLK, extractswere prepared in harsher conditions using RIPA buffer (25 mM Tris-HCl,pH 7.5, 50 mM NaCl, 0.5% sodium deoxycholate, 0.5% NP-40, 0.1%SDS) and were sonicated briefly as previously described (Kim and Edery,2006). In the case of fly material, flies were collected by freezing at theindicated times in LD or DD and total head extracts prepared using eithermodified-RIPA or RIPA buffer with sonication depending on which pro-teins we sought to detect; i.e., modified-RIPA was used for dPER andTIM, whereas RIPA with sonication was used for dCLK. We also raisednovel anti-dPER antiserum in guinea pigs using the services of CocalicoBiologicals. The immunogen was the same as previously described(Sidote et al., 1998) and in this study we used the anti-dPER antibodycalled GP339, which showed the highest dPER staining intensity withlowest background (data not shown). Primary antibodies were used atthe following dilutions; anti-V5 (Invitrogen), 1:10,000; anti-PER(GP339), 1:3000; anti-HA (3F10; Roche), 1:2000; anti-TIM (TR3),1:3000 (Sidote et al., 1998); anti-dCLK (GP208), 1:3000 (Kim et al.,2007). Gels (6%) were used to resolve dCLK, dPER and TIM, and in thecase of dCLK 3– 8% Tris-acetate Criterion gels (Bio-Rad) were also used.

For immunoprecipitation, protein extracts generated from either S2cells or fly heads were prepared using modified-RIPA buffer with theaddition of Protease Inhibitor Cocktail (Roche). To the extracts, 2 �l ofanti-HA (12CA5), anti-V5, anti-PER (GP73 or GP339), or anti-dCLK(GP208) antibody was added, as indicated, and incubated with gentlerotation for 3–5 h at 4°C followed by the addition of 25 �l of GammabindG-Sepharose (GE Healthcare) with a further incubation of 1–2 h. Beadswere collected by light centrifugation and immune complexes weremixed with 30 �l of 1� SDS-PAGE sample buffer, incubated for 5 min at

95°C and the resulting supernatants resolved by immunoblotting as de-scribed above.

Quantitative real-time PCR. The relative levels of dper and tim mRNAwere measured by quantitative real-time PCR. Total RNA was isolatedfrom frozen heads using TRI Reagent (Molecular Research Center). Fivehundred nanograms of total RNA were reverse transcribed with oligo-dTprimer using amfiRivert reverse transcriptase (GenDEPOT) and real-time PCR was performed using a Corbett Rotor Gene 6000 (Corbett LifeScience) in the presence of Quantitect SYBR Green PCR kit (Qiagen).Primer sequences used here for quantitation of dper and tim RNAs wereas described by Yoshii et al. (2007) and are as follows; dper forward:5�-GACCGAATCCCTGCTCAATA-3�; dper reverse: 5�-GTGTCATTG-GCGGACTTCTT-3�; tim forward: 5�-CCCTTATACCCGAGGTGGAT-3�; tim reverse: 5�-TGATCGAGTTGCAGTGCTTC-3�. We also includedprimers for the noncycling mRNA coding for CBP20 as previously de-scribed (Majercak et al., 2004), and sequences are as follows; cbp20 for-ward: 5�-GTCTGATTCGTGTGGACTGG-3�; cbp20 reverse: 5�-CAACAGTTTGCCATAACCCC-3�. Results were analyzed with softwareassociated with the Rotor Gene 6000 machine, and relative mRNA levelsquantitated using the 2��Ct method.

Immunohistochemistry. Confocal imaging of adult brains was per-formed as described previously (Ko et al., 2007). Briefly, adult flies weredissected in ice-cold PBS, heads were cut open, and fixed for 1 h in 4%paraformaldehyde on ice. Subsequently heads were thoroughly washedwith PBS containing 1% Triton X-100 and brains were dissected out.Brains were moved to a blocking solution comprised of PBT solution(PBS containing 0.5% Triton X-100) containing 10% horse serum andincubated for 30 min to a few hours. Primary antibodies were directlyadded to the blocking solution and incubated overnight at 4°C. Thefollowing antibodies and final dilutions were used; anti-HA antibody(3F10, Roche), 1:100; anti-PDF antibody (C7), 1:200 (Cyran et al., 2005).Subsequently, the brains were washed with PBT, followed by blockingsolution containing secondary antibodies and incubated overnight at4°C. The secondary antibodies used were Alexa Fluor 488-conjugatedanti-rat IgG (Invitrogen) or Alexa Fluor 555-conjugated anti-mouse IgG(Invitrogen), both at a final dilution of 1:200. After several washes withPBT, brains were transferred onto slides and mounted with Vectashield(Vector Laboratories). Confocal images were obtained with a LSM700Confocal Microscope (Zeiss) and processed with ZEN LE software(Zeiss).

ResultsA small conserved region on dPER (amino acids 926-977) isnecessary for its transcriptional repressor function incultured Drosophila cellsPrior work using a simplified Drosophila Schneider 2 (S2) cellculture assay identified a region of dPER that is required forstrong inhibition of dCLK-CYC-mediated transcription, termedthe dCLK-CYC inhibition domain (CCID) (Chang and Reppert,2003). The CCID encompasses amino acids 764-1034 of dPER,which includes previously identified conserved (C3 and C4) andnon-conserved (NC3 and NC4) regions (Colot et al., 1988) (Fig.1A). To explore the possible function(s) of these regions, wegenerated a series of dPER variants wherein each region was de-leted. The four variants were named dPER(�C3) (conserved re-gion 3; aa768-842), dPER(�NC3) (non-conserved region 3;aa843-925), dPER(�C4) (conserved region 4; aa926-977) anddPER(�NC4) (non-conserved region 4; aa978-999).

We first evaluated the ability of each dPER variant to inhibitdCLK-CYC-mediated transactivation using the standard lucif-erase (luc) reporter-based assay in S2 cells (Darlington et al., 1998;Chang and Reppert, 2003; Kim and Edery, 2006). WhiledPER(�NC3) and dPER(�NC4) manifested repressor activitycomparable to that observed for wild-type dPER, removal of ei-ther conserved region resulted in severely impaired repressor ac-tivity (Fig. 1B). Variations in the protein levels of the different

14460 • J. Neurosci., October 27, 2010 • 30(43):14458 –14469 Sun et al. • dCLK Binding Domain on dPER Protein

dPER variants cannot explain the differential capabilities in tran-scriptional repression since all variants were present at similaramounts (Fig. 1C, lanes 1, 5, 9, 17, and 21). Our results indicatethat the non-conserved amino acid stretches in the CCID are notrequired for dPER’s repressor activity. With regards todPER(�C3), this deletion encompasses the major DBT bindingsite on dPER (termed dPDBD), which is required for numerousaspects of dPER metabolism and function, including its ability torepress dCLK-CYC transcriptional activity (Kim et al., 2007;Nawathean et al., 2007). Although we did not perform extensivestudies on the dPER(�C3) version, it is almost certain that theseverely impaired transcriptional repressor function is due toabolishing the dPDBD region.

To examine the role that the C4 region plays in the ability ofdPER to function as a transcriptional repressor, we first tested thepossibility that C4 is essential for the nuclear localization ofdPER. However, ectopically expressed full-length dPER ordPER(�C4) did not manifest any significant differences in sub-cellular localization in S2 cells (supplemental Fig. S1, available atwww.jneurosci.org as supplemental material), consistent withprevious results indicating that the major nuclear localizationsequences on dPER are present in the C3 region (Chang andReppert, 2003). We also considered the possibility that deletionof C4 might interfere with DBT binding to dPER leading to loss ofrepressor activity, since dPDBD is adjacent to C4 (Fig. 1A). First,we assayed DBT-induced phosphorylation kinetics with the dif-ferent dPER deletion variants. As previously shown, the induc-tion of DBT evokes progressive decreases in the mobility of full-length dPER, which are mainly or solely due to differentialphosphorylation (Ko et al., 2002) (Fig. 1C). Under our standardconditions, hyperphosphorylated isoforms of dPER are readilyobserved at 12 h post-dbt induction (Fig. 1C, lane 2) and there islittle hypophosphorylated isoforms remaining at 36 h post-dbtinduction (lane 4). For dPER(�NC3) and dPER(�NC4), time-dependent changes in the conversion of hypophosphorylateddPER isoforms to hyperphosphorylated ones were similar to thatobserved for wild-type dPER (Fig. 1C), indicating that these non-conserved regions play little to no role in the DBT-dependentglobal phosphorylation of dPER.

Although DBT induction stimulated the time-dependent ap-pearance of slower migrating isoforms of dPER(�C3) and

dPER(�C4), there was a noticeable delay.For example, little to no hyperphosphory-lated species of dPER were detected at 12 hpost-dbt induction (Fig. 1C, comparelanes 2, 18, and 22; e.g., for the mutantversions, the dPER band is tight andshows little evidence of smearing). In ad-dition, fast-migrating hypophosphory-lated versions of dPER(�C3) anddPER(�C4) were still present even afterprolonged incubation with ectopically ex-pressed dbt (Fig. 1C, compare lanes 3, 19and 23), similar to what was previouslyshown for dPER(�dPDBD) (Fig. 1C,lanes 13–16) (Kim et al., 2007; Nawatheanet al., 2007).

To test whether alterations in theability of DBT to bind dPER are linkedto less efficient hyperphosphorylationof dPER(�C4), we performed immunopre-cipitation assays. Deletion of C3 dramati-cally decreased the interaction between

dPER and DBT (Fig. 1D, top, middle, compare lanes 5 and 3; i.e.,more wild-type dPER copurifies with DBT although there ismuch less total wild-type dPER in the extract compared with themutant version), as expected based on prior work showing thatthis region contains the major binding site mediating stable in-teractions between dPER and DBT (Kim et al., 2007) (Fig. 1D,compare lanes 3 and 4). In sharp contrast, dPER(�C4) stablyinteracts with DBT (Fig. 1D, lane 7). These data strongly suggestthat unlike dPDBD’s mode-of-action, the attenuation of progres-sive hyperphosphorylation and inability to block dCLK tran-scriptional activity by dPER(�C4) are not a result of losing thecapability to stably interact with DBT. In this regard we alsonoted that although there is little accumulation of highly phos-phorylated isoforms of dPER(�C4), this version of dPER stillundergoes enhanced degradation with prolonged expression ofDBT (Fig. 1E, compare lanes 7 and 8; see also Fig. 1C, comparelanes 24 and 20). Thus, while dPER(�C3), dPER(�dPDBD) anddPER(�C4) all show defects in DBT-dependent global phos-phorylation, only the latter retains the ability to undergo signifi-cant DBT-mediated decreases in abundance (Fig. 1E, comparelanes 8 and 4, and 6). This further supports previous findings thatdPER stability is not strongly linked to global phosphorylation(Chiu et al., 2008) and indicates that the C4 region is required forDBT-dependent hyperphosphorylation in a manner unrelated topromoting strong binding with DBT. Together, our results dem-onstrate that the ability of dPER to negatively regulate dCLK-CYC-mediated transcription is based on multiple conservedsubdomains within the CCID that have distinct biochemicalfunctions.

dPER(�C4) is highly defective in binding to dCLKWe next considered the possibility that the impaired repressorfunction of the dPER(�C4) variant might be a result of deficientbinding with dCLK. Indeed, little to no dPER(�C4) copurifiedwith dCLK, in sharp contrast to wild-type dPER or dPER(�C3)(Fig. 2A). Importantly, dPER(�C4) retains normal ability tobind TIM protein (Fig. 2B). These data together with the findingthat dPER(�C4) interacts strongly with DBT (Fig. 1D) indicatesthat the inability of dPER(�C4) to stably interact with dCLK isnot due to gross conformational changes in dPER resulting fromdeletion of the C4 region. We conclude that C4 is a critical dCLK

Figure 2. dPER(�C4) interacts with TIM but not with dCLK in S2 cells. A, B, S2 cells were transiently cotransfected with differentversions of pAct-dper in combination with pMT-dClk-V5 (A), or pAct-tim-3HA (B). Expression of recombinant dCLK was induced24 h after transfection by adding 500 �M CuSO4 to the medium. Cells were harvested 24 h after induction (A) or 36 h posttrans-fection (B), and protein extracts were either directly analyzed by immunoblotting (Input) or following immunoprecipitation (IP).A, IP was performed in the presence of anti-dPER antibodies (P) or a nonspecific antibody (HA), and immune complexes analyzedfor dCLK. B, IP was performed in the presence of anti-TIM antibodies and immune complexes analyzed for dPER.


Figure 3. The timing of dPER nuclear entry in the small ventral lateral neurons of p{dper(�CBD)} flies is similar to that observed for p{dper(WT)} flies. Adult flies of the indicatedgenotypes (left of panel) were collected at the indicated times in an LD cycle and processed for immunohistochemistry followed by visualization using confocal microscopy. A, Shown arerepresentative staining patterns obtained for the small ventral lateral neurons (s-LNvs) from at least 5 flies; dPER was visualized with anti-HA (3F10) antibodies labeled with Alexa Fluor488 (green). PDF was visualized with anti-PDF (C7) antibodies labeled with Alexa Fluor 533 (Cyran et al., 2005). B, The cytoplasmic/nuclear distribution of dPER for s-LNv from eachgenotype at ZT19 and ZT20 was quantified.

Table 1. Locomotor activity rhythms of p{dper(�CBD)} flies and control p{dper(WT)} transgenic fliesa

Genotypeb Temperature (°C) Period � SEM (h) Power c Rhythmicity (%)d Number e

w 1118 18 23 � 0.75 41.8 50 32wper 01;; p{dper(WT)}(M16) 18 23.4 � 0.42 34.6 29 31wper 01; p{dper((�CBD)}(M1) 18 24.3 � 0.16 36.8 27.6 29wper 01;; p{dper(�CBD)}(F3) 18 24.1 � 0.17 60.6 93.5 31per S 18 19.2 � 0.11 49.3 38.5 26per L 18 27.5 � 0.38 33.9 21.9 32w 1118 25 23.3 � 0.08 74.9 73.3 16wper 01;; p{dper(WT)}(M16) 25 23.3 � 0.05 141.7 100 32wper 01; p{dper(�CBD)}(M1) 25 26.2 � 0.22 69.4 68.8 16wper 01;; p{dper(�CBD)}(F3) 25 26.2 � 0.15 100.1 93.1 31per S 25 19.7 � 0.81 108 74.2 32per L 25 28.1 � 0.11 116.8 89.7 32wper 01;; p{dper(WT)}(M16) 29 23.3 � 0.05 106.8 100 32wper 01; p{dper(�CBD)}(M1) 29 28 � 0.17 61.3 66.7 30wper 01;; p{dper(�CBD)}(F3) 29 27.5 � 0.07 122.4 100 32per S 29 18.7 � 0.05 74 70 30per L 29 29.9 � 0.08 98.9 96.9 32aFlies were kept at 25°C and exposed to 4 d of 12:12LD followed by 7 d of DD.bM16, M1, and F3 indicate independent transgenic lines.cMeasure of the strength or amplitude of the rhythm.dPercentage of flies with activity rhythms having a power value of �10 and a width value of �2.eTotal number of flies that survived until the end of the testing period.


binding domain on dPER, although we have not ruled out thepossibility that other regions on dPER can function as bindingdomains for dCLK. Based on these findings we refer to the regionbetween amino acids 926 and 977 as the CBD (for dCLK bindingdomain). At present it is not clear whether the C4 region on dPERis sufficient to interact with dCLK.

Flies expressing dPER(�CBD) display behavioral rhythmswith longer periodsTo investigate the in vivo significance of the CBD of dPER in clockfunction, we generated transgenic flies harboring a dper trans-gene internally deleted for this region, termed p{dper(�CBD)}.The parental wild-type dper transgene [herein termedp{dper(WT)}] used to generate the deletion variant has sequencesencoding an HA epitope tag followed by a stretch of His residuesat the carboxy terminus of the dper open reading frame, facilitat-ing purification and detection of the transgene derived dPERprotein (Kim et al., 2007; Chiu et al., 2008). Two independentlines of transgenic flies bearing the p{dper(�CBD)} transgenewere obtained and evaluated in a per-null wper 01 genetic back-ground (Konopka and Benzer, 1971). Locomotor activity rhythmswere assayed under standard conditions whereby flies were kept at25°C for 4 d of 12 h:12 h light:dark (LD) cycle followed by 7 d ofcomplete darkness to determine their free-running periods. As pre-viously shown, transgenic wper 01 flies harboring the p{dper(WT)}transgene manifest robust locomotor activity rhythms with�23.5 h periods, similar to wild-type flies (Kim et al., 2007) (Table1). In sharp contrast, although both independent lines ofp{dper(�CBD)} transgenic flies exhibit strong activity rhythms, theperiods are �3 h longer than their wild-type control counterparts.The high level of rhythmicity for p{dper(�CBD)} flies was surprising

given that both dPER(�CBD) anddPER(�dPDBD) have severely im-paired transcriptional repressor func-tions (Fig. 1 B) (Kim et al., 2007) andp{dper(�dPDBD)} flies are completelyarrhythmic (Kim et al., 2007). This in-dicates that dPER(�CBD) retains somecircadian relevant activities and furthersupports functionally distinct inhibi-tory domains within the CCID.

We also determined activity rhythmsat different temperatures. As expected,p{dper(WT)} flies exhibit behavioralrhythms with �23.5 h periods over a widerange of physiologically relevant tempera-tures (Table 1). However, behavioral rhythmsin p{dper(�CBD)} flies lengthen as tempera-ture increases, suggesting the biochemicaldefect(s) of the dPER(�CBD) protein is ex-acerbated at higher temperatures. Alterna-tively, this might reveal a more fundamentalrole for the C4 region on dPER in tempera-ture compensation

dPER(�CBD) exhibits normal timingof nuclear entry in key clock cellsIn some cases, mutations in the dpergene that lead to alterations in the pe-riod of behavioral rhythms are associ-ated with changes in the timing of dPERnuclear entry in brain clock neurons(Curtin et al., 1995). The circadian system

driving adult behavioral rhythms in Drosophila is situated in thebrain and comprised of a neural network of several anatomicallyand functionally distinct clock neuron clusters, wherein the smallventral lateral neurons (s-LNv) are central for the maintenance oflocomotor activity rhythms in constant dark conditions (for re-view, see Nitabach and Taghert, 2008). Pigment dispersing factor(PDF), a circadian relevant neuropeptide, was used as a marker toidentify the LNvs and demarcate the cytoplasm (Renn et al.,1999). Wild-type dPER manifests mixed cytoplasmic-nuclearstaining at ZT19 and ZT20, and essentially only nuclear stainingbeginning at ZT22 (Fig. 3). A similar temporal pattern was ob-served for dPER(�CBD), indicating that during a standard light-dark cycle, the C4 region has at best a minor impact on the timingof dPER nuclear entry in key pacemaker neurons (Fig. 3). Thesefindings are consistent with results obtained in cultured S2 cellssuggesting the CBD does not have a major impact on dPER sub-cellular localization (supplemental Fig. S1, available at www.jneurosci.org as supplemental material).

Quasi-normal dPER/TIM biochemical rhythms inp{dper(�CBD)} transgenic fliesAs a means to more directly probe the central clock mechanismoperating in p{dper(�CBD)} flies, head extracts were preparedand the daily biochemical cycles in dPER and TIM proteins de-termined during LD and the first day of DD (Fig. 4). AlthoughdPER(�CBD) exhibits daily changes in abundance and phos-phorylation, there are several differences when compared withthe wild-type control dPER protein. For example, peak levels ofdPER(�CBD) are 1.5- to 2-fold higher compared with dPER (Fig.4A, top, compare lanes 2 and 7; Fig. 5A). Also, in light-dark cyclesdPER(�CBD) attains peak levels between ZT20 and ZT24, while

Figure 4. Levels of dPER and TIM proteins undergo robust daily cycles in p{dper(�CBD)} flies. A–C, Adult flies of the indicatedgenotypes [above the panels; WT, wper 01;; p{dper(WT)}(M16); �CBD, wper 01;; p{dper(�CBD)}(F3)] were collected at the indi-cated ZT or CT. Head extracts were prepared and analyzed either directly by immunoblotting (A, C) or following immunoprecipi-tation (IP) (B). IP was performed in the presence of anti-dPER antibodies, and immune complexes analyzed for eitherphosphorylated S47 on dPER (pS47) or total dPER. Anti-HA (3F10) or anti-TIM (TR-3) Abs were used to visualize dPER or TIM,respectively.


wild-type dPER reaches maximal values by ZT20 (Fig. 4A, top,compare lanes 15 and 16 with 7 and 8). A similar delayed accu-mulation phase for dPER(�CBD) was also observed during thefirst day of DD (Fig. 4A, bottom, compare lanes 13 and 14 with 5and 6). These results are consistent with the longer behavioralperiods manifested by p{dper(�CBD)} flies (Table 1).

In addition to alterations in the daily abundance cycle,dPER(�CBD) exhibits less dramatic shifts in electrophoretic mo-bility during the late-night/early day when the majority of wild-type dPER is hyperphosphorylated (Fig. 4A, top, compare lanes 1to 3, and lanes 2 to 7; bottom, compare lanes 1 to 8, and 5 to 7)(see also Fig. 5B). These results are in remarkable agreement withdata obtained in cultured S2 cells showing that the DBT-mediated global phosphorylation of dPER(�CBD) is impaired(Fig. 1C). Furthermore, although progressive hyperphosphoryla-

tion is attenuated, dPER(�CBD) still exhibits a robust decliningphase in abundance during the early-to-mid day (Fig. 4A, top,compare lanes 11 and 16) (see also Fig. 5B). Thus, as in S2 cells(Fig. 1; supplemental Fig. S2, available at www.jneurosci.org assupplemental material), while the C4 region affects global phos-phorylation it has little noticeable effect on the ability of dPER toundergo DBT-mediated degradation. Indeed, prior work showedthat although global hyperphosphorylation of dPER moderatelyenhances its degradation, it is not required for the rapid decline inits levels during the late night/early morning (Chiu et al., 2008).Rather, phosphorylation of Ser47 on dPER during the late night/early day is the key phospho-signal that triggers recognition bythe F-box protein SLIMB and ultimately rapid degradation viaubiquitin-proteasome pathway (Ko et al., 2002; Chiu et al., 2008).For example, when dPER is rapidly degraded at ZT2, the intensity

Figure 5. dPER(�CBD) can interact with dCLK in the presence of TIM. A–C, Adult flies of the indicated genotypes (above the panels; jrk, dClk jrk) (Allada et al., 1998) were collected at the indicatedtimes during LD cycle (A, B) or first day of DD (C). D, Adult flies of the indicated genotypes were treated with light (LP) or maintained in the dark (ZT) for 30 min starting at ZT20. Head extracts wereprepared and either directly analyzed by immunoblotting (Input) or subjected to immunoprecipitation (IP). IP was performed in the presence of anti-dCLK antibodies, and immune complexesanalyzed for dPER, TIM, or dCLK, as indicated (right of panels). E, S2 cells were transiently transfected with pAct-dper-V5 or pAct-dper�CBD-V5 in combination with pMT-HA-dClk. In addition, somecells were additionally transfected with pAct-tim (�). Exogenous expression of dCLK was induced 24 h after transfection by adding 500 �M CuSO4 to the medium. Cells were harvested 24 h afterinduction, and protein extracts were directly analyzed by immunoblotting (Input) or subjected to immunoprecipitation (IP). The protein targeted by the antibody added during IP is indicated (leftof panel) and immune complexes analyzed for the indicated protein (right of panel).


of the phosphorylated S47 signal is clearly greater compared withthat at ZT14, even though there is less total dPER protein presentat ZT2 (Fig. 4B, compare lanes 1 and 2). Although there might besubtle differences in the efficiency of S47 phosphorylation be-tween wild-type dPER and dPER(�CBD), the results further sup-port the contention that there is little to no effect of the C4 regionon the DBT-dependent mechanism regulating dPER stability.Nonetheless, the attenuated global hyperphosphorylation ofdPER(�CBD) might contribute to its relatively higher steady-state levels observed during a daily cycle (Fig. 4 A). In addition,slightly higher peak levels of dper RNA in p{dper(�CBD)} flies(see below; see Fig. 7) might also contribute to the increasedoverall abundance of the dPER(�CBD) protein.

Alterations in the abundance profile of TIM protein fromp{dper(�CBD)} flies were consistent with those observed fordPER(�CBD) protein. Most notably, the overall TIM levels inp{dper(�CBD)} flies were 1.5- to 2-fold higher, and the timing inattaining peak levels was delayed (Fig. 4C). TIM protein is essen-tially only detected during the dark phase in an LD cycle (Fig.4C, compare top and bottom) (see also Fig. 5B), indicatingthat its light-mediated degradation is not affected inp{dper(�CBD)} flies (Ashmore and Sehgal, 2003).

Light disrupts the interaction between dPER(�CBD)and dCLKTo probe for in vivo interactions between dPER(�CBD) withdCLK, we prepared head extracts from flies collected at differ-ent times during an LD cycle and performed immunoprecipi-tation assays. In control p{dper(WT)} flies, wild-type dPERinteracts with dCLK in a time-dependent manner, with thegreatest copurification occurring during the dPER-mediatedtranscriptional repression phase between ZT20 – 4, as previ-ously shown (Lee et al., 1998; Bae et al., 2000; Menet et al.,2010) (Fig. 5A, left). To our surprise and unlike the situationwhen using S2 cells, for which we could not observe interac-tions between dPER(�CBD) and dCLK (Fig. 2), dPER(�CBD)stably copurifies with dCLK (Fig. 5A, right). While the stainingintensity of dCLK copurifying with dPER (�CBD) is less than thatobserved with the control situation, the overall levels of dCLK inp{dper(�CBD)} are also generally lower (Fig. 5A, compare bot-tom). Nonetheless, it is possible that dPER(�CBD) has a weakerassociation with dCLK compared with wild-type dPER. Intriguingly,the association of dPER(�CBD) with dCLK was only observed dur-ing the dark phase, unlike wild-type dPER (Fig. 5A, top, comparelanes 1 and 8).

To better understand how light might affect the interactionbetween dPER(�CBD) and dCLK in flies, we examined moretime points during the dark-to-light transition. Wild-type dPERis stably associated with dCLK through the late night (i.e., ZT21)and well into the morning (i.e., ZT3) (Fig. 5B, top, lanes 1– 4). Insharp contrast, there is a dramatic decrease in the interaction ofdPER(�CBD) and dCLK following the onset of light (Fig. 5B,top, compare lanes 7 and 8). This striking photic effect ondPER(�CBD)-dCLK complex formation cannot be attributed tolight-mediated changes in the levels of dPER or dCLK; e.g., inp{dper(�CBD)} flies at ZT1, the levels of dPER are higher andthose of dCLK comparable to those in p{dper(WT)} flies (Fig. 5B,compare lanes 3 and 8). Together, our data strongly suggest thatdPER(�CBD) is stably associated in a complex with dCLK duringthe dark phase, but this interaction is abruptly disengaged uponexposure to photic cues.

What could be causing the photosensitivity underlying theinteraction of dPER(�CBD) with dCLK? Given that TIM is rap-

idly degraded upon light stimulation, which is also the case inp{dper(�CBD)} flies (Fig. 5B, bottom, compare lanes 2 and 3with 7 and 8), we reasoned that TIM plays a prominent role inmediating the interaction between dPER(�CBD) and dCLK. In-deed, in daily light-dark cycles dPER(�CBD) only copurifies withdCLK-containing complexes when TIM is also present in thecomplex, unlike the wild-type situation where some dPER caninteract with dCLK during the early day even in the absence ofdetectable copurifying TIM (Fig. 5B, lanes 4 and 5). Interactionsbetween dPER(�CBD) and dCLK are observed during the “sub-jective” day in constant darkness (top, compare Fig. 5C, lane 7 to5B, lane 8), conditions wherein TIM levels do not undergo sharpdecreases (Fig. 4C, bottom). Prior work showed that TIM doesnot require dPER to stably interact with dCLK in vivo (Lee et al.,1998) (Fig. 5B). Moreover, removal of the CBD does not affectthe ability of dPER to bind TIM (Fig. 2B). We also investigatedthe interaction between dPER(�CBD) and dCLK in the middle ofnight, when dPER(�CBD) and dCLK is already associated, upongetting rid of TIM by treatment of light. With light pulses of 30min duration administered on ZT20, rapid TIM degradation isobserved (Fig. 5D, third panel, lane 3 and 6). In this condition,while wild-type dPER is strongly associated with dCLK,dPER(�CBD) is rapidly dissociated from dCLK (Fig. 5D, firstpanel, compare lane 3 and 6). Although we did not do a moreextensive kinetic analysis, 30 min of light is sufficient to abolishthe interaction between dPER(�CBD) and dCLK. Together thefindings strongly suggest that in vivo TIM is mediating the asso-ciation between dPER(�CBD) and dCLK. As a more directmeans to test this possibility we used cultured S2 cells and deter-mined the ability of dPER(�CBD) to interact with dCLK in thepresence or absence of ectopically expressed TIM. Indeed, TIMstrongly enhanced the association between dPER(�CBD) anddCLK, whereas it had little to no impact on the interaction be-tween dCLK and wild-type dPER (Fig. 5E).

Direct interaction between dCLK and dPER is not necessaryfor dPER’s repressor functionBased on the ability of TIM to mediate the interaction betweendPER(�CBD) and dCLK in S2 cells (Fig. 5E), we sought to deter-mine whether the presence of TIM could also rescue the ability ofdPER(�CBD) to inhibit dCLK-CYC-mediated transcription(Fig. 6A). As previously shown, whereas TIM stimulates dPER’sfunction as a transcriptional repressor, TIM manifests little to norepressor activity on its own (Darlington et al., 1998; Rothenfluhet al., 2000; Chang and Reppert, 2003). Remarkably, dPER(�CBD)coexpressed with TIM gained the ability to inhibit dCLK-CYCtransactivation. The repression exerted by the combination ofdPER(�CBD) and TIM was �50% less compared with that ofwild-type dPER, suggesting a partially defective repressor capa-bility. Our findings strongly suggest that TIM can act as a scaffoldto bridge the association between dPER and dCLK, enablingdPER to participate in transcriptional repression.

Nonetheless, it is also possible that TIM stimulates the tran-scriptional repressor function of dPER(�CBD) by increasing itsnuclear localization (Saez and Young, 1996; Rothenfluh et al.,2000; Chang and Reppert, 2003). To test this possibility, wesought to augment the nuclear localization of dPER(�CBD) byplacing a potent nuclear localization signal (NLS) at the C termi-nus of dPER(�CBD) and measured its repressor activity. Thisstrategy was previously shown to enhance dPER’s ability to re-press dCLK-CYC-mediated transcription in S2 cells (Chang andReppert, 2003; Nawathean et al., 2007). Despite the increasedrepressor activity of wild-type dPER containing an ectopic NLS


sequence, the presence of a NLS on dPER(�CBD) did not en-hance its transcriptional inhibition capabilities (Fig. 6B). Thus,the findings clearly indicate that TIM’s stimulatory effect on thetranscriptional repressor potential of dPER(�CBD) is not an “in-direct” effect of increased nuclear localization but is almost cer-tainly due to TIM promoting the close interaction betweendPER(�CBD) and the dCLK-CYC transcription factor.

Light evokes rapid increases in dper/tim RNA levels inp{dper(�CBD)} transgenic fliesAs a result of the rhythmic inhibition of dCLK-CYC-mediatedtranscription, the levels of dper and tim mRNAs undergo dailycycles, with peak values attained between ZT12–16 and troughamounts around ZT0 – 4 (Fig. 7A,B) (Hardin et al., 1990; Sehgalet al., 1994). Although daily oscillations in dper/tim RNA levelswere apparent in p{dper(�CBD)} flies (Fig. 7A,B), peak valueswere higher. Higher daily levels of dper/tim transcripts were alsoobserved in constant dark conditions (Fig. 7C,D). These resultssuggest that repression of dCLK/CYC transactivation viadPER(�CBD) is somewhat diminished compared with that ofwild-type dPER, in agreement with results obtained in culturedcells (Fig. 6 A). However, the quasi-normal daily cycles in dperand tim transcripts indicate that circadian auto-inhibition isquite robust in flies where the sole functional version of dPERis missing the CBD.

Despite quasi-normal daily cycles in dper/tim RNA levels, wenoted that in p{dper(�CBD)} flies the rising phases are signifi-

cantly accelerated following lights-on at ZT0, whereas the decliningphases were less affected (Fig. 7A,B). Sharp increases in eitherdper or tim transcripts were not observed for p{dper(�CBD)} fliesduring the subjective day in constant dark conditions (Fig. 7C,D).To better evaluate whether light stimulation affects the daily tra-jectory in dper/tim transcript accumulation, we measured timRNA levels in two sets of flies that following entrainment to stan-dard LD cycles were either exposed to light at ZT0 or kept in thedark (Fig. 7E,F). The results clearly indicate that exposure to

Figure 6. Ectopic expression of TIM enables dPER(�CBD) to inhibit dCLK-dependent tran-scriptional activity in S2 cells. A, S2 cells were transiently transfected in the presence (�) orabsence (�) of pMT-dClk-V5. In addition, 20 ng of pAct-dper (WT) or pAct-dper�CBD (�CBD)were transfected either singly or in combination with increasing amount of pAct-tim (20, 50,100 ng). Luc activity in the absence of transfecting pMT-dClk-V5 was set to 1, and all other valueswere normalized. B, S2 cells were transiently transfected with pMT-dClk-V5 in combinationwith increasing amount of pAct-dper (WT), pAct-dper-NLS (WT-NLS), pAct-dper �CBD (�CBD),or pAct-dper �CBD-NLS (�CBD-NLS). Luc activity in the absence of any dper-containing plas-mid were set to 100% and all other values normalized. Shown are the average values from threeindependent experiments for relative luciferase activity(error bars denote SEM).

Figure 7. The levels of dper and tim transcripts undergo robust daily cycles in p{dper(�CBD)}flies and exhibit rapid increases following light-exposure. A–H, Adult flies of the indicatedgenotypes were collected at the indicated ZT (A, B, E) or CT (C, D, F ). RNA was extracted from flyheads, and quantitative real-time PCR was used to measure the relative levels of total tim (A, C,E, F–H ) or dper (B, D) RNAs. G, H, Adult flies of p{dper(WT)} (G) or p{dper(�CBD)} (H ) flies werecollected at the indicated ZT and served as controls (dark). Light exposure was initiated at ZT21,and flies were collected 1 or 2 h after light treatment (light). RNA levels at ZT21 were set to 1 andall other values were normalized. Shown are the average values from three independent exper-iments. *p � 0.005, error bars denote SEM.


light dramatically increases the levels of tim during its risingphase, indicating that dPER(�CBD)-mediated repression ofdCLK/CYC activity is rapidly attenuated by light. We also mea-sured tim RNA levels after introduction of light to p{dper(WT)}and p{dper(�CBD)} flies for 2 h starting at ZT21 (Fig. 7G,H).Treatment of light resulted in strong induction of tim RNAin p{dper(�CBD)} flies but not in p{dper(WT)} (Fig. 7G,H).These findings are in strong agreement with our demonstrationthat TIM bridges the interaction between dPER(�CBD) anddCLK (Fig. 5), and rescues the transcriptional repressor functionof dPER(�CBD) (Fig. 6A). Thus, the light-dependent degrada-tion of TIM leads to the rapid dissociation of dPER(�CBD) fromdCLK-CYC, an event that accelerates the next round of dper/timexpression.

Direct interaction between dPER and dCLK is likely requiredfor maintenance of hyperphosphorylated isoforms of dCLKdCLK is phosphorylated in vivo (Lee et al., 1998), with the ap-pearance of hyperphosphorylated isoforms in the late night/earlyday (Lee et al., 1998; Kim and Edery, 2006; Yu et al., 2006). It isthought that dPER acts as a molecular bridge to deliver kinasessuch as DBT to dCLK (Kim and Edery, 2006; Yu et al., 2006, 2009;Kim et al., 2007), an event that might be important for repressingdCLK transactivity during the late night/early morning. To de-termine whether dPER(�CBD) might provoke time-dependenthyperphosphorylation of dCLK, we examined biochemical pro-files of dCLK in p{dper(WT)} and p{dper(�CBD)} flies. In con-trol p{dper(WT)} flies, hyperphosphorylation of dCLK wasobserved from late night to early morning as previously reported(Kim and Edery, 2006; Yu et al., 2006) (Fig. 8, compare lanes 3and 4). However, in several independent experiments, we notedthat in p{dper(�CBD)} flies slowly migrating (highly phosphor-ylated) dCLK isoforms are either absent or greatly diminished(Fig. 8A, compare lane 2 and 3, and 14 and 15). This result sug-gests that the late night/early morning-specific hyperphosphory-lation of dCLK requires direct interactions between dCLK anddPER. However, we cannot rule out alternative scenarios, such ashyperphosphorylated isoforms of dCLK are less stable inp{dper(�CBD)} flies. Nonetheless, our findings clearly indicatethat dPER function is critical in regulating the phosphorylatedstate of dCLK.

DiscussionA shared feature of eukaryotic circadian pacemaker mechanismsis that daily cycles in gene expression involve the phase-specificinteraction of one or more repressors with core clock transcrip-tion factors. Initial findings identified PER proteins in animalsand FREQUENCY (FRQ) in Neurospora as key components un-derlying the main negative feedback loops operating in their re-

spective clocks (Dunlap, 1999). Earlymodels, mostly based on work in Dro-sophila and Neurospora, suggested that thedirect binding of PER or FRQ to their rel-evant transcription factors was the bio-chemical mode-of-action underlyingthese repressors. More recent work is be-ginning to refine this view and it is nowthought that these “repressors” function,at least partly, by acting as molecularbridges to ensure the timely assemblyand/or delivery of larger repressor com-plexes that inhibit elements functioningin the positive arms of circadian tran-scriptional feedback loops (Cheng et al.,

2005; Schafmeier et al., 2005; He et al., 2006; Yu et al., 2006, 2009;Kim et al., 2007; Baker et al., 2009; Chen et al., 2009). Intrigu-ingly, PER and FRQ proteins also share another role in that phos-phorylation driven changes in their daily levels are central tosetting clock pace (Bae and Edery, 2006; Gallego and Virshup,2007; Baker et al., 2009). Thus, repressors such as PER and FRQact as a critical nexus in clock mechanisms by connectingphosphorylation-based biochemical timers to the regulation oftranscription, yielding appropriately phased daily cycles in geneexpression. How the binding of these period-setting repressors tocore clock transcription factors leads to inhibition in transactiva-tion potential is not well understood.

In this study, we identified the C4 region on dPER as the soleor major dCLK binding domain (termed CBD). Despite the in-ability of dPER(�CBD) to bind to dCLK (Fig. 2), p{dper(�CBD)}flies manifest quasi-normal feedback circuitry within the coreoscillator mechanism (Figs. 4, 7), almost certainly as a result ofTIM facilitating the close interaction of dPER with dCLK (Fig. 5),enabling temporal repression of dCLK-CYC-mediated transcrip-tion (Figs. 6, 7). Thus, TIM is a bona fide component of the in vivocircadian repressor complex, and presumably plays a role inmodulating the interaction between dCLK and dPER. Moreover,we report here that direct binding between dCLK and dPER is notnecessary for dPER’s repressor activity (Figs. 2, 5, 6), but is likelynecessary for normal hyperphosphorylation of dCLK (Fig. 8).

An approach that is providing insights into how these“phospho-timing repressor” clock proteins function is the iden-tity of regions required for promoting transcriptional inhibition.In Drosophila, early work mapped a region on dPER necessary forstrong inhibition of dCLK/CYC activity in an S2 cell transcrip-tion assay (Chang and Reppert, 2003). This region, termed theCCID domain, contains two highly conserved regions, namelyC3 and C4 (Fig. 1A). The C3 region contains the sole or majordomain on dPER required for stable interaction with DBT,termed the dPDBD, and is critical not only for hyperphosphory-lation of dPER but also for inhibiting dCLK-CYC-mediated tran-scription, despite the fact that eliminating this region does notabrogate the ability of dPER to stably interact with dCLK in vivo(Kim et al., 2007; Nawathean et al., 2007). On the other hand, thenewly identified CBD is required for the physical interaction ofdPER with dCLK (Figs. 2, 5). Thus, the CCID is comprised of atleast two distinct regions with different biochemical modes-of-action; a region required for physical interaction with dCLKand another that functions as a scaffold to promote binding ofregulatory factors that modulate the activity/metabolism ofdCLK/CYC.

An unanticipated aspect of our work is that TIM can promotethe binding of dPER(�CBD) to dCLK in a simplified cell culture

Figure 8. Little to no hyperphosphorylated isoforms of dCLK are detected in p{dper(�CBD)} flies. Adult flies of the indicatedgenotypes (top of the panel) were collected at the indicated ZT. Head extracts were prepared and analyzed by immunoblotting inthe presence of anti-dCLK antibodies. ns, Nonspecific bands; arrowheads, hyperphosphorylated isoforms of dCLK.


system and in flies, an event that rescues dPER’s repressor func-tion (Fig. 6). Earlier work suggested that TIM is dispensable forrepression of dCLK/CYC transactivity (Rothenfluh et al., 2000;Ashmore et al., 2003; Chang and Reppert, 2003). However, al-though TIM exhibits little to no repressor activity toward dCLK-CYC-mediated transcription in S2 cells, it enhances that of dPER(Darlington et al., 1998; Rothenfluh et al., 2000; Chang and Rep-pert, 2003). This enhancement was largely attributed to the factthat TIM stimulates the nuclear localization of dPER (Saez andYoung, 1996; Rothenfluh et al., 2000). Our findings suggest aphysiological role for TIM in modulating circadian transcriptionby regulating the interaction between dPER and dCLK. AlthoughdPER can bind dCLK in the absence of TIM (Lee et al., 1998,1999), it is possible that by interacting with both dPER and dCLK,TIM influences the properties of the repressor complex. For ex-ample, TIM might modulate the conformation of dPER, enhanc-ing the assembly/activity of the repressor complex. In support ofthis view, brief light stimulation in the night leads to subtle butnoticeable effects on dper/tim RNA levels that precede significantchanges in the abundance of dPER (Lee et al., 1996; Rothenfluh etal., 2000). Of particular interest, dper/tim RNA levels were in-duced by light exposure in flies expressing a dPER variant (nameddPER-�C2) missing a short stretch of conserved amino acids(515–568) (Schotland et al., 2000). Although the basis for theimpaired function of dPER-�C2 was not clear, based on ourresults it is possible that its interaction with dCLK/CYC is defec-tive in the absence of TIM.

Our findings that TIM has a more pivotal role in transcrip-tional regulation might also be relevant to a recent study suggest-ing a two-step mechanism for dPER-mediated inhibition ofdCLK-CYC activity, whereby during the first phase of inhibitiondPER is bound to dCLK at the chromatin which is followed by asecond off-DNA sequestration of dCLK by dPER (Menet et al.,2010). The switch from an on-DNA to an off-DNA mechanism isthought to occur around ZT18, around the time TIM begins toaccumulate in the nucleus (Curtin et al., 1995; Menet et al., 2010).We speculate that TIM might regulate progression from an on-DNA to an off-DNA inhibitory mechanism by modulating theinteraction between dPER and dCLK/CYC.

Although dPER(�CBD) in the presence of TIM can suppressdCLK-CYC-mediated transcription, the efficiency is lower com-pared with that of wild-type dPER (Figs. 6, 7). Several differentscenarios could account for this, including less favorable spatialalignment between dPER(�CBD) and dCLK/CYC and/or effectson the ability of dPER(�CBD) to bind and/or deliver regulatoryfactors to dCLK/CYC. Perhaps a more interesting possibility issuggested by the less extensive phosphorylation of dCLK inp{dper(�CBD)} flies (Fig. 8). Hyperphosphorylated dCLK ismainly detected in the late night/early day during times whendCLK-CYC transcriptional activity is inhibited, suggesting thathighly phosphorylated dCLK is less active (Lee et al., 1998; Kimand Edery, 2006; Yu et al., 2006; Kim et al., 2007). The absence ofhyperphosphorylated isoforms of dCLK in p{dper(�CBD)} fliessuggests that direct association between dPER and dCLK isrequired to provoke dPER-dependent dCLK phosphorylation.The lack of hyperphosphorylated isoforms of dCLK inp{dper(�CBD)} flies might also contribute to the higher overalllevels of dper/tim transcripts (Fig. 7). In this context it is notewor-thy that FRQ is thought to play a major role in repressing thepositive limb of the circadian transcriptional circuits in Neuros-pora by regulating the phosphorylated state of the WCC complex,the key clock transcription factor driving cyclical gene expressionin that system (Schafmeier et al., 2005). Future studies will be

required to better understand the biochemical function of dCLKphosphorylation.

In summary, our findings demonstrate that the direct inter-action of a key repressor to its target transcription factors is notnecessary for its ability to engage in transcriptional inhibition.Moreover, TIM can promote the close association of dPER todCLK in a manner that sustains dPER’s repressor capability,revealing a more direct role for TIM in functional interactionsbetween the negative and positive limbs of the circadian tran-scriptional feedback circuits operating in Drosophila. The dCLKinteraction domain on dPER is situated very close to the dPDBDregion that functions, at least partly, by acting as a bridge topromote close interactions between regulatory factors (such asDBT) and the dCLK/CYC complex (Kim et al., 2007). The closespacing on dPER between these two functional regions suggeststhat complex architectural constraints need to be met to assemblehighly efficient repressor complexes. It will be of interest todetermine whether other repressors, such as PER proteins inmammals and FRQ in Neursopora, also have similar spatial ar-rangements. Intriguingly, in mammals the C-terminal region ofmPER2, which is downstream of the casein kinase binding (CKB)domain, has been reported to be involved in directly binding toBMAL1 (Chen et al., 2009). Finally, our findings strongly suggestthat direct interactions between dPER and dCLK/CYC are re-quired for dCLK hyperphosphorylation. It is possible that someregulatory factors stay tightly bound to key clock repressors (suchas DBT to dPER) and thus require very close contact with centralclock transcription factors to modulate them, whereas other fac-tors are “delivered” and establishing a high local concentration issufficient to promote efficient transfer from the repressors tocircadian-relevant transcription complexes.

ReferencesAllada R, White NE, So WV, Hall JC, Rosbash M (1998) A mutant Drosoph-

ila homolog of mammalian Clock disrupts circadian rhythms and tran-scription of period and timeless. Cell 93:791– 804.

Allada R, Emery P, Takahashi JS, Rosbash M (2001) Stopping time: thegenetics of fly and mouse circadian clocks. Annu Rev Neurosci24:1091–1119.

Ashmore LJ, Sehgal A (2003) A fly’s eye view of circadian entrainment. J BiolRhythms 18:206 –216.

Ashmore LJ, Sathyanarayanan S, Silvestre DW, Emerson MM, Schotland P,Sehgal A (2003) Novel insights into the regulation of the timeless pro-tein. J Neurosci 23:7810 –7819.

Bae K, Edery I (2006) Regulating a circadian clock’s period, phase and am-plitude by phosphorylation: insights from Drosophila. J Biochem140:609 – 617.

Bae K, Lee C, Hardin PE, Edery I (2000) dCLOCK is present in limitingamounts and likely mediates daily interactions between the dCLOCK-CYC transcription factor and the PER-TIM complex. J Neurosci 20:1746 –1753.

Baker CL, Kettenbach AN, Loros JJ, Gerber SA, Dunlap JC (2009) Quanti-tative proteomics reveals a dynamic interactome and phase-specific phos-phorylation in the Neurospora circadian clock. Mol Cell 34:354 –363.

Ceriani MF, Darlington TK, Staknis D, Mas P, Petti AA, Weitz CJ, Kay SA (1999)Light-dependent sequestration of TIMELESS by CRYPTOCHROME. Sci-ence 285:553–556.

Chang DC, Reppert SM (2003) A novel C-terminal domain of DrosophilaPERIOD inhibits dCLOCK:CYCLE-mediated transcription. Curr Biol13:758 –762.

Chen R, Schirmer A, Lee Y, Lee H, Kumar V, Yoo SH, Takahashi JS, Lee C(2009) Rhythmic PER abundance defines a critical nodal point for neg-ative feedback within the circadian clock mechanism. Mol Cell 36:417– 430.

Cheng P, He Q, He Q, Wang L, Liu Y (2005) Regulation of the Neurosporacircadian clock by an RNA helicase. Genes Dev 19:234 –241.

Chiu JC, Vanselow JT, Kramer A, Edery I (2008) The phospho-occupancy


of an atypical SLIMB-binding site on PERIOD that is phosphorylated byDOUBLETIME controls the pace of the clock. Genes Dev 22:1758 –1772.

Colot HV, Hall JC, Rosbash M (1988) Interspecific comparison of the pe-riod gene of Drosophila reveals large blocks of non-conserved codingDNA. EMBO J 7:3929 –3937.

Curtin KD, Huang ZJ, Rosbash M (1995) Temporally regulated nuclear en-try of the Drosophila period protein contributes to the circadian clock.Neuron 14:365–372.

Cyran SA, Yiannoulos G, Buchsbaum AM, Saez L, Young MW, Blau J (2005)The double-time protein kinase regulates the subcellular localization ofthe Drosophila clock protein period. J Neurosci 25:5430 –5437.

Darlington TK, Wager-Smith K, Ceriani MF, Staknis D, Gekakis N, SteevesTD, Weitz CJ, Takahashi JS, Kay SA (1998) Closing the circadian loop:CLOCK-induced transcription of its own inhibitors per and tim. Science280:1599 –1603.

Dunlap JC (1999) Molecular bases for circadian clocks. Cell 96:271–290.Edery I (2000) Circadian rhythms in a nutshell. Physiol Genomics 3:59 –74.Gallego M, Virshup DM (2007) Post-translational modifications regulate

the ticking of the circadian clock. Nat Rev Mol Cell Biol 8:139 –148.Hamblen-Coyle MJ, Wheeler DA, Rutila JE, Rosbash M, Hall JC (1992) Be-

havior of period-altered rhythm mutants of Drosophila in light:dark cy-cles. J Insect Behav 5:417– 446.

Hardin PE (2006) Essential and expendable features of the circadian time-keeping mechanism. Curr Opin Neurobiol 16:686 – 692.

Hardin PE, Hall JC, Rosbash M (1990) Feedback of the Drosophila periodgene product on circadian cycling of its messenger RNA levels. Nature343:536 –540.

He Q, Cha J, He Q, Lee HC, Yang Y, Liu Y (2006) CKI and CKII mediate theFREQUENCY-dependent phosphorylation of the WHITE COLLARcomplex to close the Neurospora circadian negative feedback loop. GenesDev 20:2552–2565.

Kim EY, Edery I (2006) Balance between DBT/CKIepsilon kinase and pro-tein phosphatase activities regulate phosphorylation and stability of Dro-sophila CLOCK protein. Proc Natl Acad Sci U S A 103:6178 – 6183.

Kim EY, Ko HW, Yu W, Hardin PE, Edery I (2007) A DOUBLETIME kinasebinding domain on the Drosophila PERIOD protein is essential for itshyperphosphorylation, transcriptional repression, and circadian clockfunction. Mol Cell Biol 27:5014 –5028.

Kloss B, Price JL, Saez L, Blau J, Rothenfluh A, Wesley CS, Young MW (1998)The Drosophila clock gene double-time encodes a protein closely relatedto human casein kinase Iepsilon. Cell 94:97–107.

Kloss B, Rothenfluh A, Young MW, Saez L (2001) Phosphorylation of pe-riod is influenced by cycling physical associations of double-time, period,and timeless in the Drosophila clock. Neuron 30:699 –706.

Ko HW, Jiang J, Edery I (2002) Role for Slimb in the degradation of Dro-sophila Period protein phosphorylated by Doubletime. Nature 420:673– 678.

Ko HW, DiMassa S, Kim EY, Bae K, Edery I (2007) Cis-combination of theclassic per(S) and per(L) mutations results in arrhythmic Drosophila withectopic accumulation of hyperphosphorylated PERIOD protein. J BiolRhythms 22:488 –501.

Konopka RJ, Benzer S (1971) Clock mutants of Drosophila melanogaster.Proc Natl Acad Sci U S A 68:2112–2116.

Lee C, Parikh V, Itsukaichi T, Bae K, Edery I (1996) Resetting the Drosophilaclock by photic regulation of PER and a PER-TIM complex. Science271:1740 –1744.

Lee C, Bae K, Edery I (1998) The Drosophila CLOCK protein undergoesdaily rhythms in abundance, phosphorylation, and interactions with thePER-TIM complex. Neuron 21:857– 867.

Lee C, Bae K, Edery I (1999) PER and TIM inhibit the DNA binding activityof a Drosophila CLOCK-CYC/dBMAL1 heterodimer without disrupting

formation of the heterodimer: a basis for circadian transcription. Mol CellBiol 19:5316 –5325.

Majercak J, Chen WF, Edery I (2004) Splicing of the period gene 3�-terminalintron is regulated by light, circadian clock factors, and phospholipase C.Mol Cell Biol 24:3359 –3372.

Menet JS, Abruzzi KC, Desrochers J, Rodriguez J, Rosbash M (2010) Dy-namic PER repression mechanisms in the Drosophila circadian clock:from on-DNA to off-DNA. Genes Dev 24:358 –367.

Meyer P, Saez L, Young MW (2006) PER-TIM interactions in living Dro-sophila cells: an interval timer for the circadian clock. Science 311:226 –229.

Myers MP, Wager-Smith K, Rothenfluh-Hilfiker A, Young MW (1996)Light-induced degradation of TIMELESS and entrainment of the Dro-sophila circadian clock. Science 271:1736 –1740.

Nawathean P, Stoleru D, Rosbash M (2007) A small conserved domain ofDrosophila PERIOD is important for circadian phosphorylation, nuclearlocalization, and transcriptional repressor activity. Mol Cell Biol 27:5002–5013.

Nitabach MN, Taghert PH (2008) Organization of the Drosophila circadiancontrol circuit. Curr Biol 18:R84 –R93.

Price JL, Dembinska ME, Young MW, Rosbash M (1995) Suppression ofPERIOD protein abundance and circadian cycling by the Drosophila clockmutation timeless. EMBO J 14:4044 – 4049.

Price JL, Blau J, Rothenfluh A, Abodeely M, Kloss B, Young MW (1998)double-time is a novel Drosophila clock gene that regulates PERIOD pro-tein accumulation. Cell 94:83–95.

Renn SC, Park JH, Rosbash M, Hall JC, Taghert PH (1999) A pdf neuropep-tide gene mutation and ablation of PDF neurons each cause severe abnor-malities of behavioral circadian rhythms in Drosophila. Cell 99:791– 802.

Rothenfluh A, Young MW, Saez L (2000) A TIMELESS-independent func-tion for PERIOD proteins in the Drosophila clock. Neuron 26:505–514.

Saez L, Young MW (1996) Regulation of nuclear entry of the Drosophilaclock proteins period and timeless. Neuron 17:911–920.

Schafmeier T, Haase A, Kaldi K, Scholz J, Fuchs M, Brunner M (2005)Transcriptional feedback of Neurospora circadian clock gene byphosphorylation-dependent inactivation of its transcription factor.Cell 122:235–246.

Schotland P, Hunter-Ensor M, Lawrence T, Sehgal A (2000) Altered en-trainment and feedback loop function effected by a mutant period pro-tein. J Neurosci 20:958 –968.

Sehgal A, Price JL, Man B, Young MW (1994) Loss of circadian behavioralrhythms and per RNA oscillations in the Drosophila mutant timeless.Science 263:1603–1606.

Sidote D, Majercak J, Parikh V, Edery I (1998) Differential effects of lightand heat on the Drosophila circadian clock proteins PER and TIM. MolCell Biol 18:2004 –2013.

Vosshall LB, Price JL, Sehgal A, Saez L, Young MW (1994) Block in nuclearlocalization of period protein by a second clock mutation, timeless. Sci-ence 263:1606 –1609.

Yoshii T, Fujii K, Tomioka K (2007) Induction of Drosophila behavioral andmolecular circadian rhythms by temperature steps in constant light. J BiolRhythms 22:103–114.

Yu W, Hardin PE (2006) Circadian oscillators of Drosophila and mammals.J Cell Sci 119:4793– 4795.

Yu W, Zheng H, Houl JH, Dauwalder B, Hardin PE (2006) PER-dependentrhythms in CLK phosphorylation and E-box binding regulate circadiantranscription. Genes Dev 20:723–733.

Yu W, Zheng H, Price JL, Hardin PE (2009) DOUBLETIME plays a non-catalytic role to mediate CLOCK phosphorylation and repress CLOCK-dependent transcription within the Drosophila circadian clock. Mol CellBiol 29:1452–1458.


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